Analyzing cells immobilized in block copolymers

ABSTRACT

The present invention provides the use of a composition comprising a block polymer as a support matrix in the manipulation, processing or analysis of particles, such as cells and fluorescent beads. In a preferred embodiment, the composition exhibits gel-sol thermoreversibility, micelle formation under gelling conditions, optically compatible, controllable surfactant properties, molecular sieving properties and biocompatibility. Further aspects of the invention provide (a) a support matrix composition comprising a block polymer, fluorescent beads and/or a dye for use in the manipulation, processing or analysis of particles, (b) a multichamber plate coated in a support matrix composition and (c) kits for producing the same.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a filing under 35 U.S.C. §371 of PCT/GB2005/002603filed with the Great Britain Receiving Office of the Patent CooperationTreaty on Jul. 1, 2005, which claims the benefit of British PatentApplication No. GB 0414825.0, which was filed with the British PatentOffice on Jul. 2, 2004.

FIELD OF THE INVENTION

The invention relates to thermoreversible gel compositions and theirapplications generally in research, diagnostic and screening assays andmethodology in the use of a cell- or particle- or reagent-support matrixbased applications.

BACKGROUND OF THE INVENTION

There exists a need to make biological assays faster and simpler toperform with an overriding drive to make the processes cheaper yetmaintain accuracy and reproducibility. This is due to a rapid increasein the number of research and diagnostic molecular probes available(e.g. new fluorescent reporter molecules) and the advantages in terms ofinformation content of multiplexing such assays across a range ofinstruments. Increasingly there is a need for cell-, particle- andbead-based (and a combination of these units) assays in which thepresence, for example, of cells with certain features indicates diseaseprocesses. Similarly, the demand and evolution of rational approaches inthe search for bioactive molecules for new medicines has resulted in aneed for low cost high-through-put screening (HTS) and the developmentof cell- and molecule-based assays, tools and arrays within the field offunctional analysis.

Such assays require or are enhanced by the availability of methodologiesfor:

-   i) the manipulation of cells/particles, analytes and reagents in    liquid and gel phases for processing purposes,-   ii) the controlled delivery of fluorescent/bioluminescent molecules    to cells/particles or the retention of the    fluorescent/bioluminescent-associated properties of said    particle/cells.-   iii) the controllable immobilisation of said cells/particles for the    purpose of analysis involving light collection-   iv) the retention of cell viability and cell function for periods of    time sufficient for the purposes of an analysis.

The present invention seeks to provide means for use in suchmethodologies.

A variety of hydrogels based upon thermoreversible polymers (in liquidform at elevated temperatures but in gel form at lower temperatures) areknown, including natural gel-forming materials such as agarose, agar,furcellaran, beta-carrageenan, beta-1,3-glucans such as curdlan,gelatin, or polyoxyalkylene containing compounds.

The present invention exploits to the distinct advantages and propertiesof block polymer-based gels, such as polyoxypropylene-polyoxyethyleneblock polymer gels (PBP), which unusually undergo transition to liquidform upon temperature reduction. This property can be described as‘reversed thermosetting’.

Selected properties of the PBP preparations, such as detergentproperties and the mechanical and thermoreversible properties ofhydrogels in general, have been documented and exploited in the art.

For example, reported uses and properties of PBP preparations includethe following:

Surfactant Properties

Polyoxypropylene-polyoxyethylene block polymer (PBP) has been used as anon-ionic surfactant for detergents, dispersants, binders, stabilisers,defoaming agents, emulsifying agents to name but a few. At high aqueousPBP concentrations, beyond a transition temperature, a gel can formcomprising amphiphilic block copolymer micelles. A commercial Pluronic®preparation F-127 (PEO₉₉-PPO₆₉-PEO₉₉, with E and P being polyoxyethyleneand polyoxypropylene, respectively) has been used in gel form forpharmaceutical preparations.

Reverse Agar and Biofilms

Gel-forming formulations of PBP have been described as ‘reverse agar’ intheir use. The low temperature gelling formulation has been used tosupport the limited growth of micro-organisms in conventionalmicrobiology applications. Such Pluronic®-based hydrogels have been usedextensively to assay biocide treatments. Gel-trapped micro-organismpopulations mimic the localized high cell densities observed in biofilmsand are subject to the similar nutrient and chemical gradients foundwithin natural biofilms. Such prior art uses with respect tomicro-organisms has revealed that PBP should have low toxicity in thestable gel form.

Molecular Sieve Properties

PBP gel form has the potential to form ordered micellar structures andhas been used as a separation media for nucleic acids, indicating thecoherent movement of molecules through the gel upon the passage of acontinuous or pulsed electrical current. Microdevices have also beendesigned with sieving gels within the same device to perform separationsinvolving both single- and double-stranded DNA over distances on theorder of 1 cm. Extensive comparisons have been made to compare differentgel matrices on the basis of gel casting ease, reusability, and overallseparation performance using for example a 100 base pair double-strandedDNA ladder as a standard sample.

Hydropads

Miniature size and high sensitivity of biochips is sought indiagnostics, testing, and research in medicine, veterinary science andapplications, agriculture, toxicology, environmental monitoring,forensics etc. Three dimensional biochips comprisingnon-thermoreversible gels (hydropads) have been developed consisting ofan array of three-dimensional gel elements on the hydrophobic surface ofa microscope slide. For example, a gel-based biochip project wasinitiated in Engelhardt Institute of Molecular Biology of the RussianAcademy of Science (EIMB) in 1989 resulting in the development of the‘Immobilized Micro Array of Gel Elements’ on chip (or IMAGE chip), whichcan bear oligonucleotides, DNA, proteins, small compounds or cells fixedwithin semi spherical hydrogel pads. A simple two-step procedure hasbeen developed for the large-scale manufacture of such chips. The gelpads can serve both as support for immobilisation and as individualnanoliter test tubes to carry out various specific interactions,chemical or enzymatic reactions. Chips have been produced that containimmobilized antibodies, antigens, enzymes, receptors, and differentligands.

Cell Encapsulation

Polymeric gels have also been explored as cell encapsulation materialsfor tissue engineering. Isolated mammalian cells and tissues havecountless applications in medicine and biotechnology, yet protecting andnourishing cells either in vitro or in vivo while harvesting the desiredproducts has proven difficult.

Drug and Dye Delivery

Previous clinical applications have revealed the use of hydrogels forthe purpose of local delivery of pharmacologically active agents totissues. In contrast, previous work on the “liquid less” cell stainingby dye diffusion from gels (polyacrylamide or gelatin) has beenrestricted to the use of gel systems lacking the unique thermoreversibleproperties of PBP-based gels. Such studies have been described bySmolweski et al., 2001, Cytometry 44(4):355-60. Dye delivery to cellswas observed but required a 2- to 4-fold increase in normal stainingconcentration of DNA dyes.

SUMMARY OF THE INVENTION

The present invention provides for a general means of combining the fourmethodologies stated above by exploiting the features of block polymer(for example, based on polyoxypropylene-polyoxyethylene block polymer[PBP]) gels namely the thermoreversible (gel-sol transition enablingmanipulation; as defined below) properties, micelle formation undergelling conditions (enabling the gel to act as a support/immobilizingmatrix), optical properties (low absorbance and non-fluorescent enablinglight-based optical analyses), controllable surfactant properties(enabling modified reporter molecule delivery to cells and a means ofcell solubilisation), molecular sieving properties (providingcontrolled, i.e. modified and regulated, delivery of exogenous reportermolecules) and low toxicity (enabling live cell processing). Theinvention also provides for a general means of making assays modular byenabling the control and manipulation of cells/particles and regulatingreactant and reporter molecule access for use in such cell-based assays.Specific formulations of PBP gels would provide optimal performance fora given application or excipient property.

By ‘thermoreversible’ we refer to the property of gel formation uponraising the temperature of a PBP composition above a critical transitionpoint while a liquid or sol form of the composition exists attemperatures below that transition point.

The current invention provides means for the recovery of test inoculumswithout further trauma and the use of gel in eukaryotic systems (e.g.human and animal cells).

The molecular sieving property of block polymer gels provides a means ofthe coherent delivery and behaviour of analytes (such as molecularreporter molecules) for the purpose of sequential or controlled deliveryto a system under analysis.

Thermoreversible gelling properties of block polymer gels also avoidsthermal damage thus providing an advantageous route for cell harvestingand recovery. One strategy is to encapsulate cells in a matrix thatallows for the diffusion of small molecules to and from cells.Encapsulation offers promising results, since PBP polymers are oftenbiocompatible and provide a three-dimensional scaffolding for thesimulation of support conditions in multicellular systems. To someextent the success of cellular encapsulation depends on the cell type.The purpose of the gel is to provide a matrix-gel environment, whichallows cells, isolated from different tissue to maintain their originalcellular phenotype. In an ideal situation the gel should provide aninert environment (i.e. it does not act to stimulate or activate cellsto do something abnormal). The cells placed in such gels are able tofunction normally and perhaps overtime organize the gel with thesynthesis of secreted macromolecules to form pseudo-tissue explants. Thecurrent invention incorporates all of the required features of a cellencapsulation matrix. It allows cells to be incorporated whilst the PBPgel is in liquid form. Upon gel formation the cells become trapped. Itis possible to administer the necessary reagents whilst the gel is ineither phase thus ensuring the cells are able to absorb the necessaryagents whilst in a supported environment. The gel with the trapped cellscan be placed at a lower temperature thus allowing the cells to beextracted by transition to the liquid phase even from selected regionsof the gel thereby permitting micro-selection of cell populations withgiven characteristics without any adverse effects. The same applies toany particle fixed in this way.

Potential advantages of block polymer gels for dye delivery includetheir utility in microgravity conditions and conditions where spillageis not desirable. Thermoreversible PBP preparations containingpotentially hazardous excipients (e.g. mutagenic dyes) would have theadditional safety feature of forming a gel at skin temperatures andabove thereby, reducing diffusion-limited transdermal delivery. The safedelivery of dye molecules can provide access to a wider range ofapplications and excipients. The present invention also allows for thesolid-phasing of dye delivery systems with significant safety advantagesand for the cold formulation of preparations with thermolabileexcipients.

Thus, a first aspect of the invention provides the use of a compositioncomprising a block polymer as a support matrix in the manipulation,processing or analysis of particles. In particular, the inventionprovides the use of a composition comprising a block polymer as asupport matrix in the optical analysis of particles.

The block polymer composition is not used merely as a substrate ormedium for cell culture.

Preferably, the support matrix exhibits the following properties:

-   1. gel-sol thermoreversibility;-   2. micelle formation under gelling conditions;-   3. optical compatibility (i.e. compatible with light-based optical    assays; electromagnetic spectrum 350 to 1300 nm);-   4. controllable surfactant properties;-   5. molecular sieving properties; and-   6. biocompatibility.

It will be appreciated by persons skilled in the art that the blockpolymer composition may be used as a support matrix for any particulatematter.

In a preferred embodiment, the particles are derived from or constitutea biological sample. Preferably, the particles are cells, for examplefixed or live prokaryotic or eukaryotic cells. The cells may be adherentor non-adherent.

Advantageously, the cells are selected from the group consisting of thefollowing cell types:

-   1. Animal cells including human and mammalian cells derived as    biopsy specimens (e.g. by fine needle aspirates), as tissue    explants, as primary cultures (e.g. human skin fibroblasts), as    transformed cell lines (e.g. Epstein Barr virus transformed    lymphoblasts), as immortalized cell lines (e.g. cell lines    immortalized with human telomerase reverse transcriptase [hTERT]),    and as established tumour cell lines.-   2. Human tumour cell lines including those representing specific    sites and diseases of therapeutic, diagnostic and analytical    interest, for example: Brain Cancer, Bladder Cancer, Breast Cancer,    Colon and Rectal Cancer, Endometrial Cancer, Kidney Cancer (Renal    Cell), Leukemia, Lung Cancer, Melanoma, Non-Hodgkin's Lymphoma,    Pancreatic Cancer, Prostate Cancer, Skin Cancer (Non-melanoma),    Thyroid Cancer.-   3. Cell lines with adherent (e.g. breast cancer cell lines MCF-7) or    non-adherent (e.g. the leukemia cell line CCRF-CEM or the classical    small cell lung carcinoma cell line NCI-H69) properties.-   4. Mammalian cell lines used in functional genomics studies (e.g.    NIH 3T3 murine cell line)-   5. Human tumour cells cell lines available for the purpose of drug    screening methodologies such as those indicated in the U.S. National    Cancer Institute tumour cell line panel (ref:    http://dtp.nci.nih.gov/docs/misc/common_files/cell_list.html),    comprising but not limited to: CCRF-CEM, HL-60(TB), K-562, MOLT-4,    RPMI-8226, SR, A549/ATCC, EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23,    NCI-H322M, NCI-H460, NCI-H522, COLO 205, HCC-2998, HCT-116, HCT-15,    HT29, KM12, SW-620, SF-268, SF-295, SF-539, SNB-19, SNB-75, U251,    LOX IMVI, MALME-3M, M14, SK-MEL-2, SK-MEL-28, SK-MEL-5, UACC-257,    UACC-62, IGR-OV1, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, SK-OV-3,    786-0, A498, ACHN, CAKI-1, RXF 393, SN12C, TK-10, UO-31, PC-3,    DU-145, MCF7, NCI/ADR-RES, MDA-MB-231/ATCC, HS 578T, MDA-MB-435,    MDA-N, BT-549, T-47D, LXFL 529, DMS 114, SHP-77, DLD-1, KM20L2,    SNB-78, XF 498, RPMI-7951, M19-MEL, RXF-631, SN12K1, MDA-MB-468,    P388, P388/ADR.-   6. Human tumour cell lines selected for their functional expression    of specific molecular entities such as transporters of xenobiotic    molecules (e.g. the ABCA3 drug transporter expressing in lung cancer    lines H522M, A549, and EKVX).-   7. Human tumour cells select for their convenient performance in    gene transfer studies (e.g. U2-OS human osteosarcoma cells).-   8. Single- and multi-cellular forms of vertebrates (e.g. embryos,    larval forms or derived dissociated cell preparations of zebrafish    Danio [Brachydanio] rerio).-   9. Cell lines used in ADME/Tox (Absorption, Distribution,    Metabolism, Elimination/Toxicity) screening protocols (e.g.    hepatocyte derived cell lines such as HepG2).-   10. Embryonic stem cells derived from human or murine sources.-   11. Neurones and/or supporting cells of the central nervous system    (e.g. astrocytes, oligodendrocytes, microglia and Schwann cells).-   12. Immortal somatic cell hybrids including hybrids that secrete    antibodies (e.g. hybridomas)-   13. Yeasts (e.g. Saccharomyces cerevisiae and Schizosaccharomyces    pombe)-   14. Cells derived from plants (e.g. for the analysis of in vitro    propagation methodologies for new cultivars, rare species, and    difficult-to-propagate plants).-   15. Immune response cells (e.g. antigen presenting dendritic cells)-   16. Extra- and intra-cellular forms of animal parasites (e.g.    Plasmodium falciparum).-   17. Micro-organisms including pathogenic bacteria of diagnostic    interest (e.g. Methicillin-resistant Staphylococcus aureus).-   18. Fungi including those used in pest-control (e.g.    Entomopathogenic fungi including the genera Beauveria, Metarhizium    and Tolypocladium).-   19. Single and multicellular forms of free-living animals such the    nematode Caenorhabditis elegans.-   20. Cells derived from organotypic cultures (e.g. cell clumps,    spheroids and brain slices).-   21. Cells derived from explant material (e.g. cartilage, skin and    invertebral disc).

Conveniently, the cells are capable of expressing a fluorescentmolecule. For example, the cells may be engineered by recombinant DNAtechniques to express green fluorescent protein (GFP) and/or spectralvariants and/or stability variants thereof.

Preferably, the composition provides an inert environment for cells.More preferably, the composition is sterile prior to use.

In an alternative preferred embodiment, the particles are fluorescentbeads. Such beads may provide a particle for calibration that has aspecific size (large up to 30 μm and sub-resolution, e.g. below 200 nm),fixed amount of fluorophore, unique fluorophore spectra and mixturesthereof. Suitable beads are available from Molecular Probes(Invitrogen), Carlsbad, US (e.g. FluoSpheres™).

Advantageously, the composition enables particles to be immobilisedtherein.

It will be appreciated by persons skilled in the art that the blockpolymer composition for use in the present invention must exhibitgel-sol thermoreversibility. Preferably, the composition comprises ablock copolymer of polyoxyethylene and polyoxypropylene, such as apoloxamer.

Poloxamers are polyethylene-polypropylene glycol block polymerscontaining ethylene oxide (PEO) and propylene oxide (PPO) molesaccording to the formula (See Table 1):

-   -   (PEO).sub.a-(PPO).sub.b-(PEO).sub.c.

TABLE 1 Molecular Weights of Poloxamers Av. Values Poloxamer No.Pluronic ® Av. Mol. Wt a b c 401 4,400 6 67 6 402 5,000 13 67 13 4035,750 21 67 21 407 F127 12,000 98 67 98 331 3,800 7 54 7 333 4,950 20 5420 334 5,850 31 54 31 335 6,000 38 54 38 338 F108 15,000 128 54 128 2823,650 10 47 10 284 4,600 21 47 21 288 F98 13,500 122 47 122 231 2,750 639 6 234 4,200 22 39 22 235 4,600 27 39 27 237 F87 7,700 62 39 62 238F88 10,800 97 39 97 212 2,750 8 35 8 215 4,150 24 35 24 217 F77 6,600 5235 52 181 2,000 3 30 3 182 2,500 8 30 8 183 2,650 10 30 10 184 2,900 1330 13 185 3,400 19 30 19 188 F68 8,350 75 30 75 122 1,630 5 21 5 1231,850 7 21 7 124 2,200 11 21 11 101 1,100 2 16 2 105 1,900 11 16 11 108F38 5,000 46 16 46 Certain number of the above poloxamers are also knownas Pluronic ®, which is a brand name of BASF Corporation.

Preferred are poloxamers wherein;

-   -   a is 46 to 128;    -   b is 16 to 67; and    -   c is 46 to 128.

More preferred are poloxamers wherein;

-   -   a is 46, 52, 62, 75, 97, 98, 122 and 128;    -   b is 16, 30, 35, 39, 47, 54 and 67; and    -   c is 46, 52, 62, 75, 97, 98, 122 and 128.

Most preferably, the block polymer is selected from the followingpoloxamers with recognized capacity to form gels (seehttp://www.basf.com/static/OpenMarket/Xcelerate/Preview_cid-982931200587_pubid-974236729499_c-Article.html):

Generic name Proprietary name Poloxamer 407 Pluronic ® F127 Poloxamer338 Pluronic ® F108 Poloxamer 288 Pluronic ® F98 Poloxamer 237Pluronic ® F87 Poloxamer 238 Pluronic ® F88 Poloxamer 217 Pluronic ® F77Poloxamer 188 Pluronic ® F68 Poloxamer 108 Pluronic ® F38

In a particularly preferred embodiment, the block polymer is poloxamer407 (Pluronic® F127, BASF).

The block polymer may be prepared in any suitable aqueous medium. Forexample, the block polymer is prepared in distilled water or aphysiological buffer, such as phosphate-buffered saline (PBS).

Preferably, the composition has a pH of 7.2 to 7.4.

It will be appreciated that the block polymer should be present in thesupport matrix composition at a gelling concentration. In a preferredembodiment, the block polymer is present in the composition at aconcentration of 24% (w/v).

Advantageously, the composition is in a liquid (sol) form under chilledconditions (for example, 0 to 5° C.) and yet in a semi-solid gel form atroom temperatures and above. For example, the composition may achieve agel form at a transition temperature between room temperature and 37° C.

It will be appreciated that the transition temperature of thecomposition may be modified by altering the formulation of thecomposition, for example by changing the concentration of the blockpolymer in the composition. Alternatively, the transition temperature ofthe block polymer composition may be modified by the addition of one ormore excipients, examples of which are given in Table 2.

TABLE 2 Effect of specific additives on the sol-gel transitiontemperature for a 24% w/v preparation ofPolyoxypropylene-Polyoxyethylene Block Polymer (PBP) 407. Effect ontransition Additive temperature (° C.) 1% w/v sorbitol −1.4 5% w/vsorbitol −3.0 1% w/v hydroxyethylcellulose −0.3 5% w/vhydroxyethylcellulose −2.2 1% w/v glycerol −0.2 5% w/v glycerol −2.4 1%w/v sodium chloride −4.2 5% w/v sodium chloride −10.3 1% w/v propyleneglycol −0.8 5% w/v propylene glycol −3.4 1% w/v polyethylene glycol 4000.0 5% w/v polyethylene glycol 400 +0.2 1% w/v polyethylene glycol 2000+1.1 5% w/v polyethylene glycol 2000 +2.8

In a preferred embodiment, the composition is applied to a surface of amicroscope slide, a coverslip or a multichamber plate (e.g. a multiwellplate).

In a further preferred embodiment, the composition serves a supportmatrix for the analysis of particles involving light collection,including transmission, phase-contrast, fluorescence,fluorescence-lifetime, bioluminescence, chemo-luminescence, anisotropy,light scattering, and refractive index. For example, the composition mayserve as a support matrix for the analysis of particles by imaging,microscopy or non-imaging plate based detection platforms.

Preferably, the particles are analysed by standard fluorescencemicroscopy.

More preferably, the particles are analysed by confocal laser scanningmicroscopy, multi-photon excitation laser scanning microscopy orfluorescence microscopy in which the image data collected are subjectedto mathematical processing (including deconvolution) to providedepth-specific information.

Conveniently, the light originates the light originates from agenetically encoded construct in a cell to express a fluorescentmolecule such as cells manipulated to express a fluorescent molecule,for example green fluorescent protein and/or spectral variants and/orstability variants thereof.

In a further preferred embodiment, the composition serves as a supportmatrix for the multi-dimensional analysis of particles, for example by3D(x, y, z) imaging, time (kinetic) analysis and lamda (spectral)analysis.

Alternatively, the composition may serve as a support matrix for thekinetic analysis of particles.

In a particularly preferred embodiment of the first aspect of theinvention, analysis of the particles is performed by high throughputscreening.

In another preferred embodiment, the support matrix is for use incalibration, optical alignment or orientation in methodologies requiringthe collection of light. For example, the analysis may be forcalibration purposes, point-spread function determination and eventorientation within optical slices of two or more dimensions.

In an alternative preferred embodiment, the composition serves as aparticle mountant.

In a further preferred embodiment, the composition provides a means ofcontrolling and/or modifying access of reactants and reporter moleculesto particles.

Preferably, the composition further comprises fluorescent beads. Forexample, beads may be deposited on a surface or layer within saidcomposition.

Advantageously, the composition comprises fluorescent beads of differentsizes and/or different colours (such beads are available commerciallyfrom Molecular Probes [Invitrogen Corporation], Carlsbad, US).

Alternatively, or in addition, the composition further comprises a dye,such as a DNA fluorochrome. Suitable dyes are available commercially(for example, from Molecular Probes [Invitrogen Corporation], Carlsbad,US). Preferably, the dye exhibits cell permeant properties withexcitation and emission wavelengths in the visible range spectrum,including the near infrared. Example of suitable dyes include calcein,propidium iodide and the SYTO series of dyes.

Most preferably, the composition comprises1,5-bis{[2-(methylamino)ethyl]amino}-4,8-dihydroxy anthracene-9,10-dione(DRAQ5™; available from BioStatus Limited, Shepshed, UK) or a derivativethereof.

The composition may also further comprise one or more of the followingadditives:

-   1. a cell-fixing chemical, such as paraformaldehyde (PFA);-   2. a chemo-attractant, i.e. a chemical agent, exogenously present,    eliciting directional motility in a responsive cell;-   3. an excipient for the purpose of cell protection or biological    modification (such as a growth factor or signalling molecule);    and/or-   4. an excipient for the purpose of modifying the photophysical    and/or photochemical effects of light illumination on cells or    reporter molecules (for example, the excipient may reduce    photobleaching of fluorescent reporter molecules or enhance    photobleaching of extracellular fluorescent reporter molecules).

A second aspect of the invention provides a support matrix compositionfor the manipulation, processing or analysis of particles comprising ablock polymer together with fluorescent beads and/or a dye.

Preferably, the composition exhibits the following properties:

-   1. gel-sol thermoreversibility;-   2. micelle formation under gelling conditions;-   3. optical compatibility;-   4. controllable surfactant properties;-   5. molecular sieving properties; and-   6. biocompatibility.

Further preferred embodiments of the second aspect of the invention areas defined in relation to the first aspect of the invention.

For example, the particles may be any particles as defined above inrelation to the first aspect of the invention, for example live,non-adherent cells.

Similarly, the block polymer may be any block polymer as defined abovein relation to the first aspect of the invention, for example a blockcopolymer of polyoxyethylene and polyoxypropylene. Preferably, the blockpolymer (such as a block copolymer of polyoxyethylene andpolyoxypropylene) may be present in the composition at a concentrationof 24% (w/v).

Conveniently, the composition is applied to a surface of a microscopeslide, a coverslip or a multichamber plate (for example, a 96-well,384-well or 1536-well plate).

In a further preferred embodiment, the composition is suitable for theanalysis of particles involving light collection, for example by imaging(e.g. 3D imaging), microscopy (e.g. fluorescence microscopy) ornon-imaging plate based assays.

Preferably, the light being analysed is fluorescence, bioluminescence orchemoluminescence emissions. Most preferably, the composition issuitable for high throughput screening.

The composition according to the second aspect of the invention may besuitable for calibration, optical alignment or orientation inmethodologies requiring the collection of light. For example, thecomposition may be used for calibration, point-spread functiondetermination and event orientation within optical slices of two or moredimensions.

Alternatively, the composition may serve as a particle mountant and/ormay provide a means of controlling access of reactants and reportermolecules to particles.

In a further preferred embodiment of the second aspect of the invention,the composition comprises fluorescent beads. For example, fluorescentbeads may be deposited on surface within the composition. Preferably,the composition comprises fluorescent beads of different sizes and/ordifferent colours (i.e. fluorescent spectral properties).

In an alternative preferred embodiment of the second aspect of theinvention, the composition further comprises a dye, such as a DNAfluorochrome (for example, DRAQ5™ or a derivative thereof, availablefrom Biostatus Limited, UK).

A third aspect of the invention provides a method of making a supportmatrix composition according to the second aspect of the inventioncomprising incorporating fluorescent beads and/or dye into a blockpolymer formulation. Preferably, the method comprises dissolving a blockpolymer in distilled water or phosphate-buffered saline, sterilising thesolution formed thereby, and storing the solution at 4° C.

A fourth aspect of the invention provides a kit for making a supportmatrix composition according to the second aspect of the inventioncomprising a block polymer, fluorescent beads and/or a dye.

A fourth aspect of the invention provides a microscope slide, coverslipor multichamber plate comprising a support matrix composition as definedin any one of the preceding claims applied to a surface thereof. Forexample, the multichamber plate may be a 96-well, 384-well or 1536-wellplate. Advantageously, the support matrix composition forms anaddressable array for the purpose of mechanical delivery of analytes andsubsequent optical analyses requiring the collection of light includingtransmission, phase-contrast, fluorescence, fluorescence-lifetime,bioluminescence, chemoluminescence, anisotropy, light scattering, andrefractive index.

In a preferred embodiment of the fourth aspect of the invention, thesupport matrix composition is provided on the microscope slide,coverslip or multichamber plate in a dried form which requiresrehydration prior to use.

A fifth aspect of the invention provides a method of making a microscopeslide, coverslip or multichamber plate according to the fourth aspect ofthe invention comprising applying a support matrix composition asdefined above in relation to the first or second aspects of theinvention to a surface of the microscope slide, coverslip ormultichamber plate.

Preferably, the method further comprises dehydrating the support matrixcomposition after it has been applied to the surface of the microscopeslide, coverslip or multichamber plate.

A sixth aspect of the invention provides a kit for making a microscopeslide, coverslip or multichamber plate according to a fifth aspect ofthe invention comprising a microscope slide, coverslip or multichamberplate and a support matrix composition as defined above in relation tothe first or second aspects of the invention. Preferably, a multichamberplate comprising 96 wells, 384 wells or 1536 wells.

A seventh aspect of the invention provides a method of staining cellscomprising covering or mixing cells to be stained with a support matrixcomposition according any to the second aspect of the invention.Preferably, the staining method permits live cells to be differentiatedfrom dead (apoptotic) cells.

Additional aspects of the invention include

-   1. Use of polyoxypropylene-polyoxyethylene block polymer (PBP) at    gelling concentrations (and at gelling temperatures) as an optically    compatible means of trapping and immobilising particles for the    purpose of calibration, optical alignment and/or orientation in    methodologies requiring the collection of light (including    fluorescence, fluorescence-lifetime, bioluminescence,    chemiluminescence, anisotropy and light scattering).-   2. The use of polyoxypropylene-polyoxyethylene block polymer (PBP)    at gelling concentrations as an optically compatible means of    trapping and immobilising live and/or fixed cells for the purpose of    analysis in methodologies requiring the collection of light    (including fluorescence, fluorescence-lifetime, bioluminescence,    chemiluminescence, anisotropy and light scattering).-   3. Use of a polyoxypropylene-polyoxyethylene block polymer (PBP) at    gelling concentrations as an over-layering mountant for adherent    cultures or planar preparations of live or fixed cells, for example    to provide protection and/or a controlled environment by temperature    change and gel concentration.-   4. A method for the preparation of particles, beads or cells    comprising centrifugation of the particles, beads or cells from an    aqueous suspension into a polyoxypropylene-polyoxyethylene block    polymer (PBP) gel phase within the same container.-   5. A method for sequential live cell-lysed cell analysis in situ    comprising immobilising live cells in    polyoxypropylene-polyoxyethylene block polymer (PBP) at gelling    concentrations and then diluting to impart surfactant properties to    the PBP in order to lyse the cells.-   6. A composition for in situ fixing, immobilisation/structure    support and cell staining comprising    polyoxypropylene-polyoxyethylene block polymer (PBP) at gelling    concentrations and a cell fixing chemical and/or a dye.-   7. Use of a polyoxypropylene-polyoxyethylene block polymer (PBP) at    gelling concentrations for the preparation and immobilisation of    encapsulated cells on porous or non-porous surfaces for the purpose    of short term cultivation and or a sequential analysis in which the    location of the sample is recognized for data linkage purposes.-   8. Use of a polyoxypropylene-polyoxyethylene block polymer (PBP) at    gelling concentrations for the preparation and immobilisation of    encapsulated cells on porous or non-porous surfaces for the purpose    of short-term cultivation and or a sequential analysis in which the    location of the sample is recognized for data linkage purposes.-   9. Use of a polyoxypropylene-polyoxyethylene block polymer (PBP) at    gelling concentrations for the preparation of encapsulated cells or    particles for the purposes of sample protection, manipulation or    analysis.-   10. Use of a polyoxypropylene-polyoxyethylene block polymer (PBP) at    gelling concentrations for the controlled carrier and delivery of    molecules to cells or particles by passive diffusion or    electrophoresis for the purpose of a controlled analysis    methodologies.-   11. Use of a polyoxypropylene-polyoxyethylene block polymer (PBP) at    gelling concentrations for the thermally controlled presentation of    cells or particles to a surface.-   12. A method of preparation of Polyoxypropylene-Polyoxyethylene    Block Polymer (PBP) as defined above in which the gel form is    prepared, deposited at known volumes and by mechanical means into    the wells of multi-well plates and subsequently de-hydrated to    provide for storage or transport. In a preferred embodiment the    multi-well plates will comprise 96-well, 384-well, or 1536-well    formats.-   13. A method of preparation of Polyoxypropylene-Polyoxyethylene    Block Polymer (PBP) as defined above in which the gel form is    prepared, deposited at known volumes and by mechanical means onto a    surface such as glass and subsequently de-hydrated to provide for    storage or transport. In a preferred embodiment the pattern of PBP    deposits would form an addressable array for the purpose of    mechanical delivery of analytes and subsequent optical analyses    requiring the collection of light including transmission,    phase-contrast, fluorescence, fluorescence-lifetime,    bioluminescence, chemoluminescence, anisotropy, light scattering,    and refractive index.-   14. A method of preparation of Polyoxypropylene-Polyoxyethylene    Block Polymer (PBP) as defined above in which the gel form is    prepared as in claim 20 and subsequently re-hydrated by the    introduction of appropriate volumes water or aqueous solutions of    user-specified solutes. In a preferred embodiment the multiwell    plates will comprise 96-well, 384-well, or 1536-well formats.-   15. A method of preparation of Polyoxypropylene-Polyoxyethylene    Block Polymer (PBP) as defined above in which the gel form is    prepared as in claim 20 and subsequently re-hydrated by the    introduction of appropriate volumes of aqueous suspensions of    particles or live cells or fixed cells for the purpose of optical    analyses requiring the collection of light including transmission,    phase-contrast, fluorescence, fluorescence-lifetime,    bioluminescence, chemoluminescence, anisotropy, light scattering,    and refractive index. In a preferred embodiment the multiwell plates    will comprise 96-well, 384-well, or 1536-well formats.-   16. Method of sample preparation using    Polyoxypropylene-Polyoxyethylene Block Polymer (PBP) as defined    above for the purpose of controlled re-hydration of particles or    live cells or fixed cells in with the process of re-hydration    results in a stratification of the particles or live cells or fixed    cells aiding the process of optical analysis by increasing their    frequency within a given optical plane    Properties and General Claims for PBP in Thermoreversible Gels for    Cell and Particle/Bead Preparation, Manipulation, Processing and    Analysis Using Fluorescence-Based Technologies

The current claims arise from the unique combinations of block polymerproperties for novel applications. These properties are thermoreversiblegel-sol formation where sol formation is favoured at low temperatures,particle/cell immobilisation, low toxicity, optical compatibility,molecular sieving and surfactant. The rapid formation of a gel at thetransition temperature reduces the surfactant properties of aqueous PBPproviding an immobilising and support matrix for the manipulation,analysis or processing of live cells. In a preferred embodimentresearch, diagnostic and screening assays using biological samples whichneed to be immobilised during continuous or periodic analyses bymicroscopy or imaging methods thereby reducing the compromising effectsof: cell motility, cell detachment from a substrate, the effects ofBrownian motion, physical disturbance of cell locations or the loss ofinter-relationships during sample manipulation. Live cell compatibleimmobilisation methodologies are vital for the sequential imaging ofdifferent optical planes for 3D re-construction or acquisition of imageswith time for kinetic analyses using laser scanning and camera-basedmicroscopy approaches. PBP gel properties can be modified by formulationproviding block polymers with different transition temperatures suitablefor different applications.

Protocol Overview and General Considerations

The invention relates to the use of a cell- or particle- orreagent-support/embedding matrix based upon aqueous formulations of athermo-reversible gel comprising in a preferred embodimentPolyoxypropylene-Polyoxyethylene Block Polymer (PBP) providingadvantageous properties for manipulation, processing and analysis. Thefollowing protocols describe typical methods for the preparation ofthermoreversible gels exemplified with Pluronic® F-127. F-127 (F-127polyol manufactured by BASF Corp., NJ) powder can be dissolved in 1×buffer (e.g. exemplified here using phosphate buffered saline) indeionised water at around 1-4° C. The low temperature of 4° C. isnecessary because both block copolymers can be readily dissolved inaqueous media at that temperature. This results in a homogenoussolution, mainly consisting of unimeric molecules. For example a 21.2%(w/v) solution of F-127 in 1× buffer has a low viscosity at temperaturesapproximately 4° C., at which the fluid can be manipulated for exampleby pressure, centrifugation etc. In this fluid form cells or particlescan be introduced at low temperature preserving cell function orparticle integrity without the thermal shock potential of using gelswhich only become liquid at elevated temperatures (>37° C.). In thefluid form cell and particle mixing can be achieved simply. In the fluidform other excipients, such as dyes (fluorescent probes) and reportermolecules, can be introduced to generate homogeneous preparations.Recovery of cells or particles from the liquid phase, or from dilutionsof the gel in chilled buffers, can be achieved by conventional methodsincluding centrifugation methods, filtration or magnetic separation.

At room temperature (above 15° C. selected by formulation to achieve agel form at room temperature to 37° C.), the middle P block of F-127copolymer becomes hydrophobic. The viscosity suddenly increases and thesystem becomes gel-like and provides an immobilising phase. Pluronic®F-127 in a low concentration (non-gelling) solution has both non-ionicdetergent properties and dispersive properties (“Intracellular ionactivities and membrane transport in parietal cells measured withfluorescent dyes.” Negulescu P A, Machen T E. Methods Enzymol 192, 38-81(1990)) which may not always be acceptable for cell handling, especiallyin studies in which detergent-lipid interactions may influence cellularmembrane parameters.

The PBP gel form can act as a support matrix to immobilise cells orparticles for example to reduce movement for the purposes of imaging.Immobilisation is important to allow for the inspection of high densityand information-rich fields (e.g. counting of cell/particle subsetsmarked by different fluorescent dyes or features), the imaging ofchanges in cell/particle features with time, the multiplex analysis ofcells/particles that require sequential acquisition, the imaging ofasynchronous events in fields of cells/particles, and the highresolution imaging of sub-cellular events which could be compromised ifthe cell itself was mobile. Gel formation also reduces the delivery rateof dye molecules, for example to embedded cells, and therefore providesan element of control which can be exploited to enhance or extend theform or dynamic range of an assay (e.g. separating fast and slowstaining populations with a convenient analysis timescale within adiffusion-limited system) and to permit the manipulation of dyepreparations in higher viscosity and hence safer formulations (e.g.reducing the rate of aerosol formation or of transdermal delivery inlaboratory accidents).

FIG. 1 shows a typical PBP viscosity-temperature profile indicating theparameters and the range of values that are pertinent to the utilisationof a thermo-reversible formulation for cell/particle manipulation anddye delivery. The sol-gel transition temperature (t50%) is a measure ofthe temperature at which to half the maximum gel viscosity is attained.The values t25% and t75% refer to the temperature at 25 and 75% of themaximum viscosity respectively. In addition to the measurement of thesevalues, the pH of the formulation (e.g. as adjusted to pH 7.2-7.4through the use of phosphate buffered saline as the buffer) is importantfor cell viability.

The handling concept behind the gel is that it would be stored routinelyin the cold (providing a liquid reagent form). The liquid form ismanipulated cold but forms a gel within seconds as the temperature isincreased. Controlling the warming process will provide for differentmicellar qualities in terms of the degree of ordering. The rapiditypermits incorporation into rapid assays. The instantaneous conversionfrom sol-to-gel phase provides a route for incorporation into assays.The liquid form can be used to trap, support, over-layer, or suspendparticles, beads, cells etc prior to or during manipulation (e.g.resuspend a cell pellet). Upon temperature shift (e.g. positive orpassive warming) the gel stiffens providing a cell/particle mountant.

General methodologies can be described which provide for applications inwhich live or fixed cells, particles or beads can be incorporated intothe gel with formulations which may include informative dyes or otherreporter molecules. These basic protocols can be adapted for specificapplications in candidate product screening in drug discovery, cell- andparticle/bead-based biotechnologies and numerous applications in imagingand microscopy and non-imaging plate based assays.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a typical PBP viscosity-temperature profile indicating theparameters and the range of values that are pertinent to the utilisationof a thermo-reversible formulation for cell/particle manipulation anddye delivery.

FIG. 2 shows camera images of EGFP-associated fluorescence in U2-OShuman tumour cells held in gel, demonstrating the maintenance ofcellular integrity and EGFP expression in the cytoplasm (arrow).(Panels: a, cell in full culture medium; b-d, cells overlayered with geland imaged at 0 [b], 10 [c] and 60 [d] min at 37° C.

FIG. 3 shows camera images of EGFP expressing U2-OS human tumour cells.mounted in gel following exposure to the nuclear locating fluorescentdye DRAQ5 (a: b)

FIG. 4 shows camera images of DRAQ5 in gel stained SU-DHL-4 cells heldin gel.

FIG. 5 shows laser scanning microscopy detection of cell nuclei andimmunostaining of a cell surface antigen for fixed cells supported ingel (bar=10 μm). Left panel show transmission image, middle panel showsgreen Alexa 488-NCAM immunofluorescence, and the right panel showsfar-red DRAQ5 nuclear fluorescence.

FIG. 6 shows the steps in a simple protocol to mount a sample pre-mixedin gel onto a standard microscope slide.

FIG. 7 shows the steps in modified protocols for the preparation ofsamples in gel on slides and multi-well systems.

FIG. 8 shows time-lapse imaging of beads in PBS or gel reveals efficienttrapping of fluorescent objects for sequential image collection.

FIG. 9 shows the effects of a magnetic field on dispersed magnetic beadsin a 24% PF-127 gel at room temperature.

FIG. 10 shows typical results for calcein loaded cells (MCF-7 humanbreast carcinoma cells cultured using routine methodologies inglass-bottomed chambers and using a camera-based system.

FIG. 11 shows a wide-field (CCD-camera) focus series through a 170 nmbead mounted in PF-127 24% w/v in water (inverted contrast). The slidewas mounted onto a Nikon fixed stage upright microscope, and imagedusing a x40 ELWD NA 0.6 air objective lens (pixel resolution of 0.23μm). In fluorescence mode (470/40 excitation and 525/50 emission) afocus series was collected using z-steps of 0.15 μm a total of 51 planeswere captured which is an equivalent of 7.5 μm total distance. A singlebead was cut out of the total stack and was montaged to show thediffraction rings. An image of a sub-resolution fluorescent bead (i.e.smaller than about 200 nm) showed an airy disk consisting of a centralspot surrounded by faint light and dark rings.

FIG. 12 shows the maximum projection of a focus series through a 170 nmbead mounted in PF-127 24% w/v in water (inverted contrast). Theconditions were identical to those described above. The beads remainstationary throughout the entire series which took approximately 3minutes to collect. Each bead consists of a bright (black) centre andrings around the centre; showing that each of the beads is stationary.

FIG. 13 shows two typical wide field point spread functions (PSF)obtained by resampling the data in xz. The asymmetric image arises dueto spherical aberrations (i.e. an air lens (refractive index lookinginto a 24% PF-127 gel sample refractive index 1.357). This is a typicalsituation in high content screening instruments screens where air lensesare used routinely, while the live sample sits in gel within a multiwellplate. The PSF sits at a slight slant due to the fact that the alignmentof the instrument is slightly out and off axis. Taken together the beadimages provide a quantitative evaluation of the instrument performancein conditions identical to those used for a typical live cell multi-wellimaging setup. Immobilising beads in media or physiological buffer onlyfor this kind of evaluation would not be possible.

FIG. 14 shows a comparison of the kinetics of uptake of DRAQ5 dye intoU2-OS human tumour cells held in PBS or gel

FIG. 15 shows differential staining of live and dead (arrowed) human Bcell lymphoma cells viewed by transmission (panel a) or fluorescence ofnuclei of cells stained with propidium iodide.

EXAMPLES Example I Methodological Aspects

A Typical Protocol for the Preparation of Aqueous Sterile PF-127Poloxamer Solutions

-   i) Aqueous poloxamer solutions were prepared on a percentage weight    in volume basis, by the cold process similar to that described by    Schmolka in 1972 (Schmolka, I. R. (1972) Artificial skin I.    Preparation and properties of Pluronic® F-127 gels for treatment of    burns. J. Biomed. Mater. Res. 6, 571-582.). PBP is added slowly to    distilled water and stirred constantly. The sol is thoroughly mixed    and stored at 4° C. until required.-   ii) PF-127 (e.g. batch number WPDL-510B) was obtained from BASF    Corporation (Preston, Lancashire, UK). PF-127 solutions used in cell    mountant protocols are prepared using, for example, phosphate    buffered saline (PBS). Different formulations of PBS can be used.    Typical formulations for Phosphate-Buffered Saline are:-   a. PBS as a 1× liquid, pH: 7.4±0.05 (Potassium Phosphate monobasic    (KH₂PO₄) 1.06 mM, Sodium Chloride (NaCl) 155.17 mM, Sodium Phosphate    dibasic (Na₂HPO₄-7H₂O) 2.97 mM)-   b. Dulbecco's Phosphate-Buffered Saline (D-PBS) (1×) liquid    containing calcium and magnesium (Calcium. Chloride (CaCl₂) (anhyd.)    0.901 mM, Magnesium Chloride (MgCl₂-6H₂O) 0.493 mM, Potassium    Chloride (KCl) 2.67 mM, Potassium Phosphate monobasic (KH₂PO₄) 1.47    mM, Sodium Chloride (NaCl) 137.93 mM, Sodium Phosphate dibasic    (Na₂HPO₄-7H₂O) 8.06 mM). [REFERENCE: Dulbecco, R. and Vogt,    M., (1954) Plaque formation and isolation of pure lines with    Poliomyelitis viruses. J. Exp. Med., 98:167].-   iii) PF-127 solutions requiring steam sterilisation are transferred    to 100 mL glass bottles, autoclaved at 120° C. for 20 minutes (USP,    XX11NFXV11) and subsequently stored at 4° C. until required. Solid    PF-127 requiring dissolution in D-PBS or a buffer of choice such as    RPMI culture medium (either alone, fully supplemented or    supplemented with glutamine and antibiotics) is weighed under    aseptic conditions and added to the sterile medium without mixing    and stored at 4° C. for 12 hours. After this period, any clumps of    PF-127 remaining are dispersed under aseptic conditions using a    sterile spatula and the mixture stored for a further 24 hours at    4° C. until PF-127 hydration was complete as judged by the presence    of a transparent solution (as defined by reference to refractive    index).-   iv) The presence of heat labile components in the buffer used in any    cell culture experiments may prevent steam sterilisation of PF-127    hydrated in such media. Instead, immediately prior to use the PF-127    solutions can be filter sterilised (0.2 μm pore size filters). This    approach also permits the preparation of thermolabile excipients, a    procedure not possible with dissolution in gels requiring heating to    achieve liquid form.-   v) Over-strength PF-127 solutions are used to dissolve excipients,    for example drug stock solutions, such that upon mixing the required    concentration of a dye (e.g. 20 μM DRAQ5™ or 1 μg/mL propidium    iodide) and PF-127 gel was obtained.    B Typical Step-Wise Protocol for the Physical Handling of PBP Gel    (Exemplified Here as a 24% w/v Preparation of PF-127 in PBS) for its    Use as a Cell/Particle Mountant-   i) Cell preparations are made by a standard cell culture method of    choice, including: using attached cells growing on a microscope    slide surface (e.g. a chamber slide or multichamber plate) or on a    coverslip (e.g. coverslip culture), or deposited upon a microscope    slide (e.g. by smear formation or droplet delivery or    cyto-centrifugation).-   ii) The PBP gel is prepared in a convenient container. Here a    dropper-bottle preparation is described for cells physically mixed    into the PBP gel or for cells deposited on the surface of a    microscope slide or growing on a coverslip).-   iii) Remove the PBP gel dropper bottle from the 4° C. refrigerator    (store upright overnight at 4° C. prior to use, and try not to    introduce bubbles into the liquid form when using the dropper) and    place it on crushed ice to maintain the PBP gel as liquid form and    to further chill the glass dropper inside the bottle.-   iv) Take a glass microscope slide (room temperature), place it on a    flat surface and quickly use the dropper to deposit one drop of PBP    gel into the centre of the slide. Return the dropper to the chilled    bottle immediately. The gel will rapidly stiffen on the surface of    the microscope slide. Do not touch.-   v) Take a standard coverslip (room temp) and gently/evenly place it    on top of the central mound of gel without pressing or trapping air    at the point of contact. The coverslip will appear as a “hat”    balancing on the gel.-   vi) Place the microscope slide on a bed of ice (or preferably onto a    flat metal plate on a bed of ice or a Peltier device to provide a    convenient chilling surface).-   vii) Watch the gel carefully and within seconds the gel will undergo    reverse transition and become a liquid, spreading as a mountant    under the coverslip.-   viii) When gel spreading has occurred, remove the slide from the    chilling plate and place the underside of the slide in contact with    a warming surface, for example a palm of the hand. The gel will    stiffen quickly, and retain the coverslip in place even at room    temperature. The slide can be inverted without movement of the    coverslip. The gel can be removed from the surface by irrigation    using chilled water or buffer.-   ix) With practice the deposition of the correct amount of PBP gel    onto the slide, the application of the coverslip and the sequence of    temperature shifts can produce a mounted sample in 30 secs with    perfect filling of the coverslip and no trapped bubbles.-   x) The preparation is then analysed by standard microscopy methods.    C Typical Protocol for the In Situ Staining of Live Cells at Room    Temperature Using an Aqueous Sterile PF-127 Poloxamer Solution    Prepared in Phosphate Buffered Saline at 24% w/v for the Purpose of    Staining Nuclear DNA-   i) An over strength aqueous poloxamer solution were prepared on a    percentage weight in volume basis as described and mixed with a    concentrated stock solution of the DNA dye DRAQ5™ to yield a final    concentration of 20 μM DRAQ5™ in 24% PF-127.-   ii) Using an ice-chilled pipette a 4° C. solution of DRAQ5™/PF-127    is over layered quickly onto a cell monolayer culture (e.g. human    osteosarcoma cell line U2-OS growing in a chamber slide), obtained    using standard cell culture methods. Prior to over layering the gel    the culture medium is removed and the monolayer washed using chilled    phosphate buffered saline and the chamber slide placed on a chilled    surface.-   iii) A coverslip is then placed onto the over layered gel and the    mounting procedure completed as described above.-   iv) The preparation is then analysed by standard fluorescence    microscopy methods to examine nuclear morphology of the cells as    they in situ stain with the DRAQ5™/PF-127 preparation.    D Typical Protocol for the In Situ Staining of Live Cells at Room    Temperature Using an Aqueous Sterile PBP Solution Prepared in PBS at    24% w/v for the Purpose of Distinguishing Live and Dead (Apoptotic    Cells) Using Differential Staining by Propidium Iodide-   i) An over strength aqueous PBP solution was prepared on a    percentage weight in volume basis as described and mixed with a    concentrated stock solution of the viability dye propidium iodide to    yield a final concentration of 1 μg/mL in propidium iodide in 24%    PF-127).-   ii) Using an ice-chilled pipette a 4° C. solution of PI/PBP solution    is mixed with a high-density suspension of cells for analysis (e.g.    human B cell lymphoma cell line growing as a suspension culture),    obtained using standard cell culture methods. The chilled, mixed    sample is pipetted onto a chilled microscope slide and a coverslip    added as described above.-   iii) The preparation is then analysed by standard fluorescence    microscopy methods to examine the presence of rapidly stained cells    showing abnormal nuclear morphology (apoptotic or necrotic) or cells    resisting staining representing those with intact plasma membranes.    Here trapping in the cell permits the kinetics of staining to be    observed and permits repeated analysis of a field of immobilised    cells, which would normally be lost in an image/microscopy, based    assay.-   iv) Cell samples may be pre-stained with propidium iodide in aqueous    suspensions prior to transfer to an aqueous PBP solution for example    the transfer of samples initially prepared for flow cytometry and    subsequently analysed by imaging in gel.    E Typical Protocol for the Preparation of Fluorescent Cells (e.g.    Expressing Green Fluorescent Protein) in PBP Gel for Live Cell    Imagine-   i) Cells carrying a fluorescent reporter are prepared using standard    cell culture methods either as attached cultures or resuspended    cells at high density in a medium of choice.-   ii) For attached cell cultures, PBP gel in liquid phase is    over-layered as described above.-   iii) For cell suspensions, aliquots are mixed directly into the PBP    gel in liquid phase and pipetted directly onto a microscope slide    with a coverslip added as described above.-   iv) The live cell preparations are then analysed by standard    fluorescence microscopy methods to examine features of interest.

Additional applications of the invention include the following:

Polyoxypropylene-Polyoxyethylene Block Polymer (PBP) at gellingconcentrations can be used to act as an optically compatible means oftrapping and immobilising particles for the purpose of calibration,optical alignment and orientation in methodologies requiring thecollection of light including fluorescence of bioluminescence emissions.In a preferred embodiment fluorescent beads deposited on a surfacewithin a PBP gel would be used in fluorescence microscopy systems (e.g.confocal laser scanning microscopy system or multi-photon excitationlaser scanning microscopy) to provide a means of calibration,point-spread function determination and event orientation within opticalslices two or more dimensions.

Calibration samples include the co-mixing of beads with cells within thePBP gel to provide a depth versus fluorescence correction versusscattering for the determination of point spread function in the samelive sample conditions. Such samples may also be used to provide anindication of performance of optical elements or instrument set-up. Sucha method would be appropriate for any type of multi-dimension imagingwhich requires calibration of x, y or z-axis resolution. Calibration isrequired in order to measure and consequently correct for sample derivedaberrations. Embedded beads co-mixed with the cellular sample aretherefore appropriate for multi-dimensional resolution measurementparticularly x,y,z axis resolution, including the point spread functionobtained from sub-resolution beads. Other aberrations require depthdependent correction of fluorescence, fluorescence spectral overlap andcross talk measurement.

Polyoxypropylene-Polyoxyethylene Block Polymer (PBP) at gellingconcentrations can be used to act as an optically compatible means oftrapping and immobilising live and fixed cells for the purpose ofanalysis in methodologies requiring the collection of light includingfluorescence or bioluminescence emissions. The cells may be non-adherentor processed cell suspensions. In a preferred embodiment thefluorescence would originate from a fluorescent molecule manipulated tobe expressed by the cell such a green fluorescent protein (GFP).

Polyoxypropylene-Polyoxyethylene Block Polymer (PBP) at gellingconcentrations as an over-layering mountant for adherent cultures orplanar preparations of live or fixed cells providing a convenientmountant for protection of cells and in situ staining or labelling ofcells. Here the sol-gel transition as a function of temperature providesa novel means of spreading the mountant at lower temperature andcontrolling the gel depth by halting spreading through gel formation byraising local temperature of the preparation. The adherent propertieswould allow for inversion of a mounted specimen so that invertedmicroscopy formats can be used. Here the gel provides an aqueous-gelphase between the specimen and another optical interface for imaging. Ina preferred embodiment the fluorescence would originate from afluorescent molecule manipulated to be expressed by the cell such agreen fluorescent protein (GFP).

Polyoxypropylene-Polyoxyethylene Block Polymer (PBP) at gellingconcentrations can be used in a method of preparation of particles,beads or cells (‘analytes’) by the centrifugation from aqueoussuspension into a PBP gel phase within the same container. In apreferred embodiment the PBP gel is present below an over-layeringaqueous phase comprising a suspension of said analytes and maintains agel-aqueous interface by temperature control. Centrifugation forcesentry of analytes into the gel. Analytes deposited into the gel phasecan be recovered by temperature-controlled transition to a sol followingremoval the aqueous over layer.

Analytes can be pre-labelled with fluorescent or bioluminescent probes.Additionally analytes which are fluorescent or bioluminescent molecularprobes may be present either in the aqueous phase or in the gel phase toenable an optical analysis of the suspended particles, beads or cells.In a preferred embodiment the fluorescent molecular probe is theanthraquinone DRAQ5™.

Polyoxypropylene-Polyoxyethylene Block Polymer (PBP) at low non-gellingconcentrations has surfactant properties which can provide celldisrupting or lytic properties for the release of molecules for primaryand/or secondary analyses. Modulation of properties would require ashift in concentration of PBP by in situ dilution and or a shift intemperature. In a preferred embodiment PBP gels solubilised in situwould impart surfactant properties and provide for a sequential livecell-lysed cell analysis methodology.

Polyoxypropylene-Polyoxyethylene Block Polymer (PBP) at gellingconcentrations can be combined with cell fixing chemicals (e.g.paraformaldehyde) and or dyes (e.g. a DNA fluorochrome) to provideunique multi-functional agents for in situ fixing,immobilisation/structure support and cell staining. In a preferredembodiment such multi-function agents would reduce processing time,minimise cell loss through a reduction in the number of processing steps(e.g. in fixation schedule that require washing and fluid removal steps)and provide a means for maintaining osmotic environments, metabolicgradients and structural/mechanical integrity.

The formation of Polyoxypropylene-Polyoxyethylene Block Polymer (PBP)gels to enable the preparation and immobilisation of encapsulatedprokaryotic cells on porous or non-porous surfaces for the purpose ofshort term cultivation and or a sequential analysis in which thelocation of the sample is recognised for data linkage purposes. In apreferred embodiment temperature-shifting the low temperature liquidphase encapsulation of a prokaryotic cell(s) could be used to trap cellsat a specific location at which a drug can be delivered for the purposeof chemosensitivity testing.

The formation of Polyoxypropylene-Polyoxyethylene Block Polymer (PBP)gels to enable the preparation and immobilisation of encapsulatedeukaryotic cells on porous or non-porous surfaces for the purpose ofshort term cultivation and or a sequential analysis in which thelocation of the sample is recognised for data linkage purposes. In apreferred embodiment temperature-shifting the low temperature liquidphase encapsulation of a eukaryotic cell(s) is used to trap cells at aspecific location at which a subsequent analysis of a gene sequence(s)and or protein(s) or other cell-originating molecules.

The formation of Polyoxypropylene-Polyoxyethylene Block Polymer (PBP)gels to enable the preparation and immobilisation of encapsulated cellson porous or non-porous surfaces for the purpose of short termcultivation and or a sequential analysis in which the location of thesample is recognised for data linkage purposes. In a preferredembodiment temperature-shifting the low temperature liquid phaseencapsulation of a eukaryotic cell(s) is used to trap cells at specificlocations for the purpose of detecting and analysing the presence orabsence of parasites including the intracellular forms of Plasmodiumspecies in the diagnosis of malaria and for the purpose of species andvariant identification.

The formation of Polyoxypropylene-Polyoxyethylene Block Polymer (PBP)gels to enable the preparation of encapsulated cells or particles forthe purposes of sample protection, manipulation or analysis. In apreferred embodiment the low temperature liquid phase encapsulation of acell or particle permits the generation of droplets for the purpose ofpreparing arrays or replicates through the delivery of such droplets toa receiving surface or container prior to or following analysis ofinformative features of the encapsulated sample.

A methodology to provide a means of the pre-building of modular assaysystems/devices for sequential processing regulated by the properties ofthe thermoreversible gels. In passing through the transitiontemperature, for example at the point of droplet formation or delivery,encapsulated samples would suffer reduced evaporation stress for livecell preparations but have increased surface adhesion properties. In apreferred embodiment encapsulated cells offer a physical protection forcells from mechanical stress imparted by sorting and arrayinginstrumentation.

The rapid formation of the Polyoxypropylene-Polyoxyethylene BlockPolymer (PBP) gel provides initially an immobilising layer on the cells.With the addition of potential chemo-attractants within the gel or in alayer above the gel, this gradient becomes an active layer forstimulating cells or attracting/sorting cells away from unstimulatedcounterparts. The thermoreversibility allows these cells to beselectively removed and further processed.

The micelle environment of the Polyoxypropylene-Polyoxyethylene BlockPolymer (PBP) provides for the controlled carrier and delivery ofmolecules (e.g. reactants, reporter fluorochromes or conjugates thereof)to cells or particles by passive diffusion or electrophoresis for thepurpose of a controlled analysis methodologies. In a preferredembodiment the molecular sieve effects of the PBP gel would effect asequential delivery of reactants and fluorescent or bioluminescentreporter molecules within sample preparations.

The addition of excipients for the purpose of cell protection orbiological modification would impart additional functionalities toPolyoxypropylene-Polyoxyethylene Block Polymer (PBP) gels. For example,the inclusion of growth factors or signalling molecules to maintain ormodify specific cellular phenotypes.

The addition of excipients for the purpose of modifying thephotophysical and photochemical effects of light illumination on cellsor reporter molecules would impart additional functionalities toPolyoxypropylene-Polyoxyethylene Block Polymer (PBP) gels. For example,excipients may be included to reduce the photobleaching of fluorescentreporter molecules.

The formation of Polyoxypropylene-Polyoxyethylene Block Polymer (PBP)gels to enable the thermally controlled presentation of cells orparticles to surfaces, which enhance or enable assay performance. In apreferred embodiment the assay would exploit surface plasmon resonanceeffects or light collection from highly restricted depths at opticalinterfaces.

The block copolymer relevant to this invention may comprisepolyoxyethylene and polyoxypropylene. Accordingly, gel-formingpreparations include those described as Pluronics® F127, F108, F98, F87and F88 (Pluronic® is a registered trademark of BASF Corporation).

Example II Use of Block Polymer Compositions of the Invention in theAnalysis of Fluorescent or Dyed Cells

1. General Methods for the Preparation of a Cell Line Expressing EGFPand its Optical Analysis

Preparation of construct. The cell cycle phase marker DNA construct (GEHealthcare; Cardiff UK) was prepared from three DNA fragments that werefused in frame and cloned into a pCI-Neo (Promega) vector that had beencut with BglII and NheI to remove the CMV promoter. The three fragmentsused were the cyclinB1 promoter, the N-terminal 171 amino acids of thehuman cyclin B1 coding region and EGFP. The cyclin B1 promoter wasamplified from a construct described previously22 using PCR and theprimers 5′-CGCGGCAGCTGCCCGAGAGCGCAGGCGC-3′ and5′-CGCAAGCTTCCTCTTCACCAGGCAGCAGCTC-3′. The N-terminal region of cyclinB1 mRNA, encoding the cyclin B1 destruction box and the CRS butexcluding the CDK binding site was amplified with HindIII and BamHI endsusing PCR and the primers 5′-GGGAAGCTTAGGATGGCGCTCCGAGTCACCAGGAAC-3′[SEQ ID NO:1] and 5′-GCCGGATCCCACATATTCACTACAAAGGTT-3′ [SEQ ID NO:2]from a cyclinB1 cDNA described previously5. The gene for EGFP wasamplified from pEGFP-N2 (Clontech) with primers5′GGTACGGGCCGCCACCATGGGATCCAAGGGCGAGGAGCTGTTCAC [SEQ ID NO:3] and5′-GGTACGGGTTAACCGGTCTTGTACAGCTCGTCCATG 3′ [SEQ ID NO:4].

All three fragments were fused and the integrity of the final cloneconfirmed by sequence analysis.

Cell reporter system. The parental cell line used in these studies was ahuman osteosarcoma cell line derived from a 15 year old Caucasian femaleU-2 OS (American Type Culture Collection [ATCC] HTB-96). U-2 OS cellswas transfected with the cell cycle marker DNA construct using Fugene(Roche) according to the manufacturers instructions. Following selectionwith 1000 μg/ml Geneticin (Sigma G7040) the expressing cells wereenriched using high-speed fluorescence activated cell sorting (MoFlow;DAKO-Cytomation) and sorted into 96 well plates (1 green fluorescentcell/well). Colonies were expanded and clones whose green fluorescencevaried with the cell cycle as predicted for a cyclin-based reporter, asdetermined by conventional flow cytometry, were expanded and a highexpressing subline maintained.

Growing and maintenance condition. The stably transfected cells weremaintained at 37° C. and 5% CO2 using standard tissue culturetechniques. Media used was McCoys 5A modified (Sigma) supplemented with2 mM glutamine, 100 units/ml penicillin, 100 mg/ml streptomycin, 10%fetal calf serum and 1000 μg/ml geneticin.

Time-lapse/camera imaging. High resolution fluorescence cell trackingwas performed with cells seeded into 8 well Nunc coverglass chambers(Labtek Inc). Culture dishes were placed on to a time-lapse instrumentdesigned to capture bright-field phase images and GFP fluorescence(480/25 nm excitation and 525/30 nm emission). An Axiovert 100microscope (Carl Zeiss, Welwyn Garden City, UK), was fitted with anincubator for 370 C/5% CO2 maintenance (Solent Scientific, Portsmouth,UK), and an ORCA-ER 12-bit, CCD camera (Hamamatsu, Reading, UK).Illumination was controlled by means of a shutter in front of thetransmission lamp, and an x, y positioning stage with separate z-focus(Prior Scientific, Cambridge, UK) controlled multi-field acquisition.Image capture was controlled by AQM 2000 (Kinetic Imaging Ltd). Allimages were collected with a 40×, 0.75 NA air apochromat objective lensproviding a field size of 125×125 mm. Sequences were captured asrequired. When required analysis of the images was performed with theintegrated AQM 2000 software package (Kinetic Imaging Ltd). Each cell inthe field was tracked individually. Fluorescence tracking on a singlecell basis was achieved in Lucida (KI Ltd). Fluorescence was recorded ina region of interest.

2. Typical Analysis of Fluorescent Cells in Gel.

FIG. 2 shows typical results for EGFP-associated fluorescence inexpressing cells imaged in culture medium (panel a), in which the markedregion shows the presence of a group of 3 cells expressing high levelsof EGFP in the cytoplasm (arrow). Following capture of the image cells,culture medium was aspirated and the cell monolayer overlayered with achilled (4° C.) 24% w/v gel (PF-127; in sol form) prepared in PBS,returned to the imaging platform (at 37° C.; the overlayer forming asupporting gel at this temperature) and the location of the fieldre-found. Further fluorescence images were captured of the same cells at0, 10 and 60 min incubation in gel. The images clearly show themaintenance of cellular integrity (flattened cells) and GFP expressionin the cytoplasm.

3. Typical Analyses of More than One Fluor in Live Cells in Gel:EGFP-Expressing Cells in Gel (24% w/v PF-127 Prepared in PBS) Co-Stainedwith a DNA Dye (DRAQ5).

FIG. 3 shows typical results for EGFP-associated fluorescence in 3samples stained for nuclear DNA using the fluorescent agent DRAQ5.Paired images show DRAQ5-associated far-red fluorescence (FIG. 3 leftpanels a, c and e) or EGFP green fluorescence (FIG. 3 right panels b, dand f). Panels a and b show results for the same cells stained in PBSwith DRAQ5 (20 μM×10 min) and imaged in PBS permitting theidentification of cells (presence of a nucleus; arrowed) withhigh-(hgfp) or low-(Igfp) EGFP expression within the cytoplasm(arrowed). Panels c and d show cells also pre-stained using DRAQ5 in PBSbut overlayered with gel (see above) after aspiration of the DRAQ5solution. The images in panels c and d show the continued ability todistinguish hgfp and lgfp expressing cells. Panels e and f show cellsstained with DRAQ5 in the gel overlayer for 1 h (DRAQ5 20 μM in 24% w/vPF-127 prepared in PBS; at 37° C.), demonstrating the ability todistinguish hgfp and lgfp cells using an in-gel staining methodology.The images clearly show the maintenance of cellular integrity (flattenedcells) and GFP expression in the cytoplasm.

4. Typical Light Transmission and Fluorescence Analysis of Cells Stainedin Gel Using a Cell Permeant Dye (DRAQ5).

Human B cell lymphoma cells (line SU-DHL-4) were cultured in suspensionusing routine methodologies. Cell culture typically contain live cellsand a background of dying cells, debris and occasionally non-cellularparticles. In a typical analysis to distinguish objects a comparison canbe made of transmission and fluorescence images. A typical methodologywould comprise cell samples pre-mixed with cooled gel (24% w/v PF-127prepared in PBS and containing 20 μM DRAQ5) and mounted under acoverslip on a cooled microscope slide. The slide was then raised toroom temperature for 30 min to permit the continued in-gel staining ofnuclear DNA by DRAQ5. FIG. 4 shows a typical field imaged fortransmission (panel a) or far-red fluorescence of a DNA dye (DRAQ5; bluelight excitation panel b). The images (see arrows) reveal the positivein-gel staining of intact cells, permitting the distinction ofbi-nucleate objects (i.e. dividing cell), debris (indistinct nuclearsignal) or non-cellular (non-DNA-containing) inclusions. The analysisexemplifies the imaging of non-adherent cells/objects, held in gel,enabling the sequential examination of cell/object features without lossof location in 3-dimensions.

5. Examples of the Use of Block Polymer Compositions of the Inventionfor Immobilizing Non-Adherent Cells for the Use High Resolution Imagingto Determine Immunofluorescence Localization.

An important feature of the PF-127 gel formulations is that they providean easy method for immobilizing suspension cells such as those preparedfor flow cytometry. This enables high resolution imaging to be performedon cells that are not originally tethered to an optical surface.

Therefore PF-127 formulations provide a route for interfacing differentcytometry platforms (e.g. a flow cytometry sample analysed by imaging)particularly those that require the sequential analysis of cells insuspension. Of particular interest is the localization of a givenfluorescence signal to a cellular compartment (e.g. the expression ofthe neural cell adhesion molecule [NCAM] on the cell surface of smallcell lung carcinoma cells [SCLC cells]) or the expression of a signal inrelationship to neighbouring cells where a support matrix is required tomaintain a cellular cluster during, for example, multiple optical scansof a confocal or multiphoton microscope.

Here the use of gel as a support matrix for fixed cells probed with anappropriate fluorescently-tagged antibody and a DNA stain is described.NCI-H69 cells were cultured as suspension cells in RPMI-1460 culturemedia with 10% FCS using standard cell culture methodologies. Cells wereharvested and fixed in ice-cold methanol for 20 minutes. After washingin phosphate buffered saline the samples were processed for standardimmunofluorescence as used for flow cytometric analysis and fluorescencemicroscopy. These suspensions were prepared as flow analysis for NCAM(CD-56) detection, using mouse anti-human (CD-56; BD Pharmingen, UK)monoclonal antibody, followed by a secondary staining using ananti-mouse Alexa 488 (Molecular Probes, InVitrogen, USA). Finally thepreparations were labelled with DRAQ5 to distinguish the nucleus.

A small sample of cells (50 μl at 1×106 cells per ml) was placed in achamber coverslip (Nunc) and PF-127 sol at 24% w/v in PBS was placedover the cell layer, and left at room temperature to form a gel layer(see part A for chamber slide preparation). The cells and cell clustersbecame immobilized under the gel matrix.

High resolution confocal laser scanning microscopy (BioRad 1024 MP;BioRad Microscience Ltd) was performed to obtain a three channel imageof the cell clump (FIG. 5). The transmission image showed opticalcompatibility with 488/647 nm light. The sample stability enabledimaging of the tightly coupled cells and provided distinct edges betweencells depicting NCAM localization. Nuclear localization depicted thecellular localization and clearly shows the number of cells within theclump. There were no detectable background or optical scatteringproblems associated with the gel mountant. The example demonstrates theuse of the gel with fixed cell preparations, within a protocolcompatible with flow cytometric analyses and the ability high resolutionimmunofluorescence signals in gel.

Example III Examples of the Production of a Microscope Slide orMultiwell Plate Coated in a Block Polymer Composition

1. Simple Protocol to Mount a Sample Pre-Mixed in Gel onto A StandardMicroscope Slide (FIG. 6 Panels a-e)

-   Step a: a Sample for Analysis is Mixed into Gel (in Sol Form; Held    in a Sample Tube on ice) for example by the addition of a    concentrated suspension of cells (e.g. 4×10⁵ cells in a 10 μl volume    of PBS prepared using standard centrifugation methodology) to a 250    μl volume of 24% w/v F-127 prepared in PBS). Over-strength    preparations of gel can be used to provide a final concentration of    24% w/v F-127 prepared in PBS if required.-   STEP b: The sample is quickly streaked across the surface of a    standard microscope slide at room temperature and the gel stiffens    within seconds.-   STEP c: A coverslip is placed onto the gel.-   STEP d: The slide is placed on an ice-pack and the gel transformed    to a liquid state and spreads under the coverslip within seconds.-   STEP e: Removing the slide from the ice-pack results in air-warming    of the slide to room-temperature and the setting of the gel within    seconds.    2. Modified Protocol to Mount a Sample(s) in Gel at Given Locations    on a Standard Microscope Slide (FIG. 7 Panels a-k)

Pre-prepare stained or unstained cells, beads or particles in an aqueoussuspension, aspirate supernatant and hold pellet on ice. FIG. 2 (panelsa-k) shows the subsequent steps for preparation of a single sample on amicroscope slide, the procedure being repeated for multiple samples asrequired.

-   STEP a: Press a silicon isolator (type shown in panel a is a S2560    silicon isolator with 8 holes [each 2 mm deep, 9 mm diameter];    obtained from Sigma-Aldrich UK) onto a microscope slide on an    ice-pack.-   STEP b: Add 90 μL of cold 35% PF-127 gel into a well. This can be    achieved, for example, using a pre-chilled 1 ml micropipette tip.-   STEPS c and d: Inject the cell/bead or particle sample through the    gel at the bottom of the well in a volume of 10 uL and the    suspension plumes to the central area of the surface of the gel.-   STEP e: Transfer slide to a warm heating block (held at 37° C.) and    the gel stiffens.-   STEP f: Carefully peel of the isolator.-   STEP g: The gel disk revealed is self-supporting.-   STEP h: Place slide onto an ice-pack until the bottom surface of the    gel starts to liquefy.-   STEP i: Place a coverslip onto the surface of the gel disk while the    slide remains on the ice-pack. The disk continues to liquefy and    starts to spread.-   STEP j: Overlayer the slides with an absorbent paper and gently    press to complete spreading of the sample and to remove excess    liquid.-   STEP k: Return to warm heating block to stiffen gel and complete    preparation. Return to room temperature for storage (e.g. up to 24    h).    3. Modified Protocol to Mount a Sample in a Chamber/Well (e.g.    Standard Glass Bottom 8 Well Chamber Slide) (FIG. 7 Panels l & m)

Pre-prepare stained or unstained cells, beads or particles in an aqueoussuspension, aspirate supernatant and hold pellet on ice. The procedurefor the addition of the gel and sample is reversed from that describedabove. FIG. 2 shows the main steps of introducing the cell/bead orparticle sample in a volume of 10 μL into an empty well/chamber of amulti-chamber slide held on an ice-pack (panel l). Then add 90 uL ofcold 35% PF-127 gel into the well (panel m). This can be achieved, forexample, using a pre-chilled 1 ml micropipette tip. The liquid geloverlayers the sample suspension. The slide is then warmed (e.g. on aheating block at 37° C.) to stiffen the sample-gel interface, asdescribed above, prior to analysis.

4. Simple Exemplar Protocol for the Preparation of Dried Films of BlockPolymer and their Reconstitution in a Multi-Well Plate (Table 3)

An example of a methodology is described for the preparation of driedfilms of PF-127 and their reconstitution by the addition of differingvolumes of water (or a given solution) to provide a range of potentialgel/liquid concentrations for cell/bead or particle immobilisation ormanipulation. The steps are outlined below.

-   STEP a: A 19.3% w/v PF-127 solution in water was prepared. This    concentration permits a liquid state to be easily formed when    chilled (e.g. at 4° C.) but still retain some degree of loose    gel/liquid state at room temperature (20° C.).-   STEP b: Volumes of cold gel were dispensed into a matrix of 48 wells    within a standard 96-well (flat bottomed) transparent plastic dish    as indicated in the Table.-   STEP c: The plate was held on a heating block at 37° C. for 24 h to    allow for the desiccation of the gel into dried films covering the    base of each well. Here the process may be accelerated for example    by vacuum drying.-   STEP d: At this stage the dried films can be stored before    commitment to rehydration.-   STEP e: Volumes of ice-cold PBS are dispensed into each well as    indicated in the Table and the plate rotated briefly to aid the    wetting of the dried. Here the process may be accelerated by    mechanical vibration.-   STEP f. The plate is then held with the lid sealed for 24 h at 4° C.    Here the rehydration conditions may be varied (e.g. incubating at    37° C. in a humidified atmosphere).-   STEP g: After rehydration the plate is returned to room temperature    for the assessment of quality gel formation in the wells by direct    and microscopic examination of the transparency and the mechanical    properties by agitating the well contents with a pipette tip.

Results: Table 3 shows the ability to prepare liquid and gel-like phasesin all combinations when assessed at room temperature. Herereconstitution was achieved using PBS demonstrating the in situpreparation of gels with a buffer of choice. Some wells showing liquidphase (i.e. ‘liquid’) at room temperature would be capable of forminggels if the temperature was raised. Further, only some combinationsresulted in the formation of a transparent and optically acceptable gel(i.e. ‘transp. gel’) with in other case a turbid-opaque gel/paste formed(i.e. ‘gel’). The combination of a 75 μL 19.3% PF-127 dried gel filmreconstituted with a 50 μL PBS volume provided a transparent gel(reversible to a sol by chilling) with a nominal poloxamer concentrationof 29%. This preferred combination would allowing for the retention ofroom temperature (and 37° C.) immobilisation properties and permit thefurther addition of sample volumes upon (for example reducing the finalconcentration of gel to 24%).

TABLE 3 Reconstitution of dried films of PF-127 (19.3% w/v gel in water)and the effects of reconstitution with differing volumes of PBS μL gelDried gel reconstituted with PBS (μL) dried: 10 25 50 100 150 200 10trans. gel liquid liquid liquid liquid liquid 20 trans. gel trans. gelliquid liquid liquid liquid 25 trans. gel liquid liquid liquid liquidliquid 50 trans. gel liquid liquid liquid liquid liquid 75 trans. geltrans. gel trans. gel liquid liquid liquid 100 gel trans. gel trans. gelliquid liquid liquid 150 gel gel gel liquid liquid liquid 200 gel gelgel trans. gel liquid liquid

Example IV Example of the Production of a Block Polymer CompositionComprising Fluorescent Beads and/or a Dye

1. Simple Protocol to Prepare Fluorescent Beads in a Gel for the Purposeof Immobilisation and Analysis

Analysis of beads may, for example, require the determination of beadlocation and optical properties such as fluorescence. An example of atypical protocol for the analysis of fluorescence characteristics andbead location is shown in FIG. 8 for red-fluorescent approx. 1 μmdiameter beads (e.g. Becton Dickinson Calbrite APC beads; BDBiosciences, USA) using excitation and emission conditions described bythe manufacturer. The general methodologies for preparing beads in gelhave been described above. A concentrated preparation of beads (e.g. 1drop into 0.5 ml gel and mixed on ice using a pipette micro-tip. A gelsample was prepared on a microscope slide. A time-lapse imaging systemdescribed above was used to sequentially image the fluorescence of beadseither in PBS (as a film trapped under a coverslip) or in 24% w/v gel(PF-127 prepared in PBS). Images a to d shows the same field of view forbeads in PBS, imaged 4 times with a 1 sec interval between each imagecapture. Image e shows the 4 merged images of a-d. Similarly, images f-ishow 1 sec interval images for beads in gel at room temperature with thecorresponding merged image shown in panel j. The beads clearly move inthe PBS preparation, due to fluid movement and Brownian motion,resulting in a confused merged image. Beads remain at fixed locations inthe gel for the scanning period demonstrating the immobilizationproperties of the gel for beads.

2. Simple Protocol for the Preparation of Magnetic Beads in Gel andtheir Manipulation in a Magnetic Field

Magnetic bead technology is in common use for separation methodologies.Unlabelled magnetic beads (obtained from The Reagent Mine Ltd., MeltonMowbray, UK; approx 2 μm diameter) were dispersed into gel (24% w/vPF-127 prepared in water) at 4° C. in a 2 mL polypropylene sample tube.The suspension was then prepared on a microscope slide for imaging bytransmitted light using a standard microscope equipped with a camerasystem. FIG. 9 (panel a) shows the dispersed beads immobilized in gel atroom temperature. A neodymium magnet (The Reagent Mine Ltd., MeltonMowbray, UK) was then placed 2.5 cm from the centre of the field of viewand the same height as the slide surface and after 30 seconds the fieldre-imaged. FIG. 9 (panel b) shows the effect of the magnetic fieldresulting in bead alignment along the lines of force, demonstrating theability to move beads in a supporting gel for the purposes of alignmentand re-location within the gel.

3. Preparation of a Dye in Gel

Vital-labelling methodologies often require the uptake of anon-fluorescent form of a dye which becomes fluorescent uponintracellular processing. Here the preparation of the vital dyecalcein-AM is described for the in-gel staining of live cellsoverlayered with the gel-dye preparation. Calcein dye (calcein AM; 0.1μg/ml; C3099 Cat. No., Molecular Probes, InVitrogen) was mixed into a24% w/v PF-127 prepared in PBS and overlayered onto a monolayer cultureof human MCF-7 cells in a chamber slide and incubated at roomtemperature for 15 min. Images were collected using standard confocalmicroscope methodologies (system: BioRad 1024 MP, BioRad Microsciences,UK). FIG. 10 shows an optical section through cells demonstrating atypical compartmentalization of the dye in some cells with more diffusestaining in others. The results demonstrate the ability to prepare a dyein gel for live cell marking and function.

Example V Examples of the Use of Block Polymer Compositions of theInvention in the Calibration of Equipment for Optical Analysis (E.G. theDetermination of Point Spread Function)

1. Gel Preparations Used for Immobilization have Advantageous

Imaging instruments (microscopes and HCS instruments) produce aspatially sampled array of fluorescence. Images may be produced by theoptical system directly (camera-based) or built up by scanning (laserscanning microscope). Fewer changes in refractive index at the differentoptical interfaces are advantageous. The availability of aqueous-basedgels provides an advantageous medium in terms of refractive index whencompared, for example, with higher RI glycerol-based mountants. Standardrefractometry was used to measure the RI values for typical gelpreparations and the values obtained are shown in Table 4.

TABLE 4 Typical values for refractive index obtained for gelpreparations Sample Refractive index (RI) PF-127 24% w/v prepared inwater; at 1.357 37° C. PF-127 24% w/v prepared in PBS; at 1.359 37° C.PBS 1.333 water 1.331

Spatial resolution in theory: Illumination wavelengths (from an arclamp) are selected by an excitation filter or spectrometer and the lightis spread onto a field aperture by a high Numerical Aperture condenserlens. It then reflects from a 45 degrees dichroic mirror and an image ofthe field aperture is demagnified into the sample by an objective lens.In this way, the entire sample is evenly bathed in light. Fluorescenceis collected by the objective and forms an image in the microscope thatis either inspected visually, using a magnifying eyepiece, or passed toan appropriate photo-detector such as a CCD camera. All parts of theilluminated sample contribute to the image that contains sharp (infocus) features as well as out-of focus features. It is important toconsider the performance characteristics of any fluorescence imaginginstruments. An image of a sub-resolution fluorescent bead (i.e. smallerthan about 200 nm) will show an airy disk consisting of a central spotsurrounded by faint light and dark rings. Measurement of the airy diskgives parameters describing the microscope performance. The distancefrom the centre to the first dark ring describes horizontal (x, y)resolution and is given by:dxy=0.61λ/NA

If a focus series of images of the bead is collected, the correspondingaxial (z) resolution is:dz=3.7dxyη/NA

-   -   η=refractive index of sample medium        -   λ=wavelength    -   NA=objective lens Numerical Aperture

Total intensity in any horizontal plane is proportional toNA2/(magnification) and is constant near the focus, so there is nooptical sectioning in a conventional microscope.

Spatial resolution in practice: The rigor in which an assay can beimplemented on any imaging system is dependent on reproducibility andcalibration of the instrument. It is essential to understand the spatialperformance of the imaging system in order to extract quantitativeinformation or indeed undertake deconvolution processing to extract 3Dinformation. Since the refractive index of the sample medium linearlyinfluences axial resolution and the axial performance changes is depthdue to spherical aberration it is important to calibrate the axialresolution ‘in situ’. The accepted method for obtaining the x,y,zperformance of a microscope is to acquire image from a sub-resolutionbead in the exact same conditions used for imaging the sample. Howeverin water-based samples (physiological buffers and media) it is essentialto keep the cells living and acquire xyz calibration information from abead. By placing the sample (cells) and beads in block polymercompositions high resolution images of immobilized beads can be obtainedenabling axial performance to be extracted.

To exemplify the use of block polymer compositions with integratedsub-resolution beads we are able to obtain information on the opticalperformance of the instrument at different depths through the sample.

Materials and Sample Preparation:

-   (i) Sub-resolution beads can be obtained from many different    manufacturers in this case Molecular Probes. PS-speck Microscope    point Source Kit: 505/515 nm fluospheres carboxylate modified    microspheres 0.17 μm yellow-green fluorescent (concentration 107 per    ml).-   (ii) Block polymer composition (PF-127) at a formulation of 24% w/v    in water was prepared as previously described above.-   Step 1: Take 0.5 mls of 24% Pluronic® F127 maintained at 4° C. and    mix with 5 μl bead solution.-   Step 2: Maintain at 4° C. on ice until ready to use-   Step 3: Place 50 μl on to a microscope slide (the droplet becomes    gel)-   Step 4: Place a coverslip (22 mm×22 mm) onto the droplet-   Step 5: Cool the slide on an ice block and the droplet spreads and    the coverslip becomes level.

Obtaining a focus series of images through the bead along the opticalaxis (see figures)

-   Step 1: Firmly secure the slide to the microscope (vibrations will    disturb image collection)-   Step 2: Choose the appropriate imaging conditions to obtain the    focus series.

Results are shown in FIGS. 11 to 13.

Reference

White N S, Errington R J. Fluorescence techniques for drug deliveryresearch: theory and practice. Adv Drug Deliv Rev. 2005 Jan. 2;57(1):17-42.

Example VI Examples of the Use of Block Polymer Compositions of theInvention in the Controlled Delivery of Reagents to Cells

1. Delivery of a Cell-Permeant DNA Dye to Cells in Gel

The delivery of reagents to cell, beads or particles immobilized in gelpermits the analysis of modified interaction kinetics over extendedperiods. Described herein is an example of the impact of gel-baseddelivery of a reagent, cell permeant DNA dye DRAQ5, in comparison withthe kinetics obtained by the staining in PBS alone. Here attached U-2 OS(American Type Culture Collection [ATCC] HTB-96) cells were grown inglass-bottomed chamber slides using standard cell culture methodologies.The use of attached cultures allowed for their immobilization forstaining in PBS and a direct comparison with the staining in gel. Theculture medium was aspirated and replaced with either PBS supplementedwith DRAQ5 (20 μM) or overlayered with gel (24% w/v PF-127 prepared inPBS) also containing DRAQ5 (20 μM). Samples were then imaged using atime-lapse microscope and the changes in nuclear associated far-redfluorescence monitored in individual cells analysed. FIG. 14 shows theuptake kinetics in PBS versus gel for individual cells. The wellrecognised asynchronous nature of cell cultures under normal growthconditions results in a range (2-fold) of cellular DNA contentsrepresenting the cell cycle age distribution of the population. In PBSthere is a rapid staining of cells with the expected spread innear-equilibrium values for nuclear fluorescence intensity. In gelstaining also re-iterates the spread in values but with slower kinetics(>10-fold) as expected from a gel-diffusion limited staining of cells.

2. Differential Staining of Live and Dead Cells in Gel Using aFluorescent Dye

In FIG. 15, propidium iodide (PI) enters into damaged cells (undergoingcell death) due to the inability of damaged plasma membranes to excludethe cationic dye. Intact healthy cells do not stain if membraneintegrity is preserved. A typical analysis for live/dead celldiscrimination in gel is described here. DoHH2 (human B cell lymphomacells) cell line has a normal background of apoptotic (dying) cells thatare normally distinguishable by there positive staining using PI. FIG. 2shows a comparison of the transmission and red-fluorescence images, uponblue light excitation, of cells held and stained in gel (24% w/v PF-127prepared in PBS; containing 1 μg/ml propidium iodide) at roomtemperature for 15 min. There is the clear ability to distinguishpositive and negative staining cells showing that gel delivery of areagent can be used for the purpose of event discrimination.

1. A method for analyzing particles comprising immobilizing theparticles in a composition comprising a block copolymer and analyzingthe immobilized particles.
 2. The method of claim 1, wherein thecomposition exhibits the following properties: gel-solthermoreversibility; micelle formation under gelling conditions;compatible with light-based optical assays in the electromagneticspectrum of 350 to 1300 nm; controllable surfactant properties;molecular sieving properties; and substantially non-cytotoxic.
 3. Themethod of claim 2, wherein the block copolymer is present in thecomposition at a gelling concentration.
 4. The method of claim 1 whereinthe particles are derived from or constitute a biological sample.
 5. Themethod of claim 1, wherein the particles are cells.
 6. The method ofclaim 5, wherein the cells are fixed.
 7. The method of claim 5, whereinthe cells are live.
 8. The method of claim 5, wherein the cells arenon-adherent.
 9. The method of claim 5, wherein the cells are selectedfrom the group consisting of human cells, animal cells, cultured celllines, immortal somatic cell hybrids, yeast cells, plant cells andfungal cells.
 10. The method of claim 5, wherein the cells are capableof expressing a fluorescent molecule.
 11. The method of claim 1, whereinthe particles are fluorescent beads.
 12. The method of claim 1, whereinthe block copolymer is a block copolymer of polyoxyethylene andpolyoxypropylene.
 13. The method of claim 12, wherein the blockcopolymer is selected from the group consisting of poloxamer 407,poloxamer 338, poloxamer 288, poloxamer 237, poloxamer 238, poloxamer217, poloxamer 188 and poloxamer
 108. 14. The method of claim 1, whereinthe composition is applied to a surface of a microscope slide, acoverslip or a multichamber plate.
 15. The method of claim 1, whereinthe composition serves as a support matrix for the analysis of particlesinvolving light collection.
 16. The method of claim 15, wherein thelight is fluorescence, bioluminescence or chemoluminescence.
 17. Themethod of claim 1, wherein the composition serves as a support matrixfor the analysis of particles by imaging, microscopy or non-imagingplate based assays.
 18. The method of claim 1, wherein the analysis isfor calibration, optical alignment or orientation in methodologiesrequiring the collection of light.
 19. The method of claim 1, whereinthe analysis is for calibration, point-spread function determination andevent orientation within optical slices of two or more dimensions. 20.The method of claim 1, wherein the composition further comprisesfluorescent beads.
 21. The method of claim 1, wherein the compositionfurther comprises a dye.
 22. The method of claim 21, wherein the dye isa DNA fluorochrome.
 23. The method of claim 21, wherein the dye is1,5-bis{[2-(methylamino)ethyl]amino}-4,8-dihydroxyanthracene-9,10-dione.
 24. The method of claim 1, wherein the particlesare encapsulated in the composition comprising a block copolymer. 25.The method of claim 1, wherein the method is for multidimensionalanalysis of particles.
 26. The method of claim 25, wherein themultidimensional analysis is selected from the group consisting of3D(x,y,z) imaging, time (kinetic) analysis and lambda (spectral)analysis.
 27. The method of claim 1, wherein the analysis is performedby high throughput screening.
 28. The method of claim 1, wherein thecomposition comprising a block copolymer provides a means of controllingor modifying access of reactants and reporter molecules to theparticles.